Aspartate 55 in the Na+/proline permease of Escherichia coli is essential for Na+-coupled proline uptake.
Aspartate 55 in the Na+/proline permease of Escherichia coli is essential for Na+-coupled proline uptake.
复制标题
大肠杆菌的 Na/脯氨酸通透酶中的天冬氨酸 55 对于 Na 偶联的脯氨酸吸收至关重要。
作者：
M. Quick;H. Jung
用中性或带电氨基酸残基分别替换了大肠杆菌Na+/Pro通透酶N-末端的4个酸性残基(环2中的Asp33、Asp34和Asp55，跨膜区II中的Glu75)。取代渗透酶中唯一可能位于跨膜区中间的残基Glu75，Asp33或Asp34对Na+偶联的Pro转运动力学影响很小或没有影响。相反，去除55位的羧酸盐(Asp55-->；Asn或Asp55-->；Cys渗透酶)会完全损害对Pro的摄取，而将该位置的侧链延长一个亚甲基(Asp55-->；Glu渗透酶)则允许以较低的初始速率转运。重要的是，所有的渗透酶分子都存在于膜中，其浓度与野生型蛋白相当。对Asp55--&Gt；Glu渗透酶催化的Na+偶联脯氨酸转运的动力学分析表明，与野生型相比，Pro的K(M)增加了5倍，V(Max)降低了30倍。值得注意的是，Glu取代了Asp55，导致渗透酶对Na+的表观亲和力降低了50倍。此外，用半胱氨酸或天冬氨酸氨基转移酶替代Asp55可阻断Pro诱导的Na+摄取，而Asp55--&Gt；Glu通透酶能显著地转运Na+。此外，在低Na+浓度下，含Asp55--&Gt；Glu通透酶的失能细胞不能检测到脯氨酸向其浓度梯度下的转运。然而，在高浓度Na+存在的情况下，观察到了向下转运的活动。在测试的所有条件下，用ASN或Cys取代Asp55会损害下坡运输。观察结果表明，脯氨酸通透酶55位的羧酸盐是Na+偶联的脯氨酸转运所必需的。提示Asp55可能参与偶联离子的结合。
Four acidic residues in the N-terminal domain of Na+/proline permease of Escherichia coli (Asp33, Asp34, and Asp55 in putative loop 2, Glu75 in putative transmembrane domain II) were individually replaced with neutral or charged amino acid residues. Replacement of Glu75, the only residue in the permease presumed to be in the middle of a transmembrane domain, Asp33, or Asp34 had little or no influence on the kinetics of Na+-coupled proline transport. In contrast, removal of the carboxylate at position 55 (Asp55 --> Asn or Asp55 --> Cys permease) impaired proline uptake completely while lengthening of the side chain at this position by one methylene group (Asp55 --> Glu permease) allowed transport at a reduced initial rate. Importantly, all permease molecules were present in the membrane at concentrations comparable to the wild-type protein. Kinetic analysis of Na+-coupled proline transport catalyzed by Asp55 --> Glu permease revealed a 5-fold increase of the K(m) for proline and a 30-fold decrease of the V(max) compared to wild-type. Remarkably, replacement of Asp55 by Glu led to a 50-fold decrease of the apparent affinity of the permease for Na+. Furthermore, replacement of Asp55 with Cys or Asn blocked proline-induced Na+ uptake whereas significant Na+ transport was observed with Asp55 --> Glu permease. In addition, transport of proline down its concentration gradient was not detectable with deenergized cells containing Asp55 --> Glu permease at low Na+ concentrations. However, downhill transport activity was observed in the presence of high Na+ concentrations. Replacement of Asp55 with Asn or Cys impaired downhill transport under all conditions tested. The observations demonstrate that a carboxylate at position 55 of proline permease is essential for Na+-coupled proline transport. It is suggested that Asp55 may be involved in binding of the coupling ion.